PROJECT SUMMARY With neither vaccines nor well-tolerated therapies available, the genus Trypanosoma causes debilitating human diseases of high morbidity for which effective drugs are urgently needed. Trypanosoma cruzi causes Chagas Disease (CD) in Central and South America, resulting in 50,000 annual deaths, with >40 recent cases reported in Texas. Trypanosoma brucei rhodesiense and T. brucei gambiense cause African sleeping sickness (HAT), affecting over 60,000 people world-wide. Both CD and HAT comprise unmet medical needs for which effective medicines do not exist. The establishment and maintenance of human infection by both T. cruzi and T. brucei requires, respectively, the related cysteine proteases cruzain and TbCat B. The objective of this proposal is to identify novel reversible-covalent inhibitors (RCIs) of cruzain and TbCatB, that exert potent and selective enzyme inhibition and trypanocidal activity in cell cultures of T. cruzi and T. brucei spp., and are curative in murine models of trypanosomiasis. Our approach builds on precedent compounds such as K11777, a tri-peptide analogue that contains an electrophilic vinyl- sulfone moiety (Fig. 1 ? SPECIFIC AIMS). K11777 is an irreversible covalent inactivator of cruzain and TbCatB which forms a stable covalent adduct with the active-site cysteines of protozoal cysteine proteases. K11777 cures Chagas disease in animal models, and has progressed to human clinical trials. However, recent focus has shifted from irreversible enzyme inactivators to reversible covalent inhibitors (RCIs), as the latter have fewer toxicity issues and are more selective for their intended targets. We will examine the introduction of a 2-vinyl- heterocyclic group in a selective peptide scaffold, replacing the vinyl-sulfone ?warhead? of K11777, as we believe such a moiety would provide suitable RCIs for cruzain and TbCatB. To date we have made peptidomimetic 2-vinyl heterocycle inhibitors (P2VHIs) which include a pyrimidine, oxazole or thiazole group conjugated to the vinyl group. These P2VHIs exhibit time- dependent, reversible inhibition of cruzain (Ki = 7-20 nM), and kill trypanosomes in cell culture. We propose that (a) the electrophilicity of the vinyl group may be ?tuned? by substitution of the heterocyclic group, and that (b) addition of an enzymatic cysteine residue on the vinyl group will be readily reversible. In the R21 phase of this proposal, we will advance this concept and produce a series of P2VHIs that exhibit (a) improved potency for cruzain, (b) inhibition of TbCatB, (c) contain peptide scaffolds which are selective for protozoan proteases, and (d) be trypanocidal at low micromolar concentrations. We will determine if a reversible covalent adduct is formed with the active-site cysteines of these targets. In the R33 phase, we will develop a chemical model for the 2-vinyl heterocycles that allows us to probe and optimize the reversible reactivity of thiols toward P2VHIs. We will produce a series of second-generation P2VHIs with the physicochemical properties, toxicity and safety profiles of drug candidates. We will evaluate these compounds in cellular models of human infection, followed by proof-of-concept assessment in murine models of CD and HAT.